Hybrid biosensors

• US 6,051,422 A
• Filed: 07/09/1998
• Issued: 04/18/2000
• Est. Priority Date: 05/30/1997
• Status: Expired due to Fees

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Kovacs, Gregory T. A., Borkholder, David A.
• Primary Examiner(s): Redding, David A.
• Application Number:
  US09/113,015
• Time in Patent Office: 649 Days
• Field of Search: 435/173.1, 435/173.4, 435/285.2, 435/287.1, 435/288.7, 204/403, 356/246, 356/446, 324/447, 324/692
• US Class Current: 435/287.1
• CPC Class Codes: C12M 41/46 of cellular or enzymatic activity or functionality, e.g. cell viability
• CPC Class Codes: G01N 33/48728 Investigating individual cells, e.g. by patch clamp, voltage clamp investigating individual particles in general G01N15/10

Abstract

The invention relates to an apparatus and method for monitoring cells and to a method for monitoring changes in cells upon addition of an analyte to the cell'"'"'s environment, comprising a device which includes an array of microelectrodes disposed in a cell culture chamber, upon which array a portion of cells adhere to the surfaces of the microelectrodes. The diameter of the cells are larger than the diameters of the microelectrodes. A voltage signal is applied across each of the microelectrodes and a reference electrode. Detection and monitoring of the signals resulting from the application of the voltage signal provides information regarding the electrical characteristics of the individual cells, including impedance (combined cell membrane capacitance and conductance), action potential parameters, cell membrane capacitance, cell membrane conductance, and cell/substrate seal resistance. Such an invention is useful in detecting or screening a variety of biological and chemical agents.

Claims

1. An apparatus comprising:

a monolithic device including an array of planar microelectrodes disposed on a substrate, the array of microelectrodes being connected to a conductive pattern having contact points connected thereto;

a cell culture chamber in which the integrated device is disposed, the cell culture chamber being configured to contain a medium for cultured cells in a volume surrounding the respective surfaces of the microelectrodes, wherein cells are introduced into the cell culture chambers and a portion of the cells adhere to respective surfaces of the microelectrodes,

wherein the diameter of a cell is larger than the diameter of a microelectrode;

a reference electrode disposed within the cell culture chamber;

means electrically connected to the contact points connected to the array of microelectrodes for selectively applying an electrical signal between each of the microelectrodes and the reference electrode;

means for detecting signals resulting from the application of the signal between each of the microelectrodes and the reference electrode;

a phase detector electrically connected to the signal detecting means; and

means for monitoring characteristics of cells which adhere to the respective surfaces of the microelectrodes based on impedance values derived from the resulting signals, the characteristics being selected from the group consisting of impedance, action potential parameters, cell membrane capacitance, cell membrane conductance, and cell/substrate seal resistance.

2. The apparatus according to claim 1, wherein the respective diameters of the microelectrodes are less than or equal to one half the diameter of a cell adhered thereto.

3. The apparatus according to claim 1, wherein the respective diameters of the microelectrodes are between about 4 and 20 μ

m.

4. The apparatus according to claim 1, wherein the monolithic device includes an integrated sensor which provides an electrical signal indicative of the pH of the medium.

5. The apparatus according to claim 1, wherein the monolithic device includes a heating means formed integrally therein.

6. The apparatus according to claim 5, wherein the monolithic device further includes a thermal isolation membrane.

7. The apparatus according to claim 5, wherein the heating means comprises an integrated thin film heater.

8. The apparatus according to claim 1, wherein the monolithic device further includes an integrated temperature sensor, the temperature sensor providing an electrical signal used to selectively activate a heater means, the heater means maintaining a substantially constant temperature.

9. The apparatus according to claim 1, wherein the monolithic device further includes an integrated optical sensor.

10. The apparatus according to claim 1, wherein the characteristics comprise action potential parameters.

11. The apparatus according to claim 1, wherein the characteristics comprise impedance.

12. The apparatus according to claim 1, wherein the characteristics comprise impedance and action potential parameters.

13. The apparatus according to claim 1, wherein the characteristics comprise cell membrane capacitance.

14. The apparatus according to claim 1, wherein the characteristics comprise cell membrane conductance.

15. The apparatus according to claim 1, wherein the characteristics comprise cell/substrate seal resistance.

16. The apparatus according to claim 1, further including means for measuring action potential of cells adhered to the surface of the microelectrodes, wherein the impedance is determined substantially simultaneously with measuring action potential.

17. The apparatus according to claim 1, wherein the cells include cells differentiated from primordial stem cells.

18. The apparatus according to claim 1, wherein the means for monitoring detects activation of voltage-gated ion channels of the cells adhering to microelectrodes on the basis of the characteristics.

19. The apparatus according to claim 1, wherein the means for monitoring detects changes in characteristics associated with the introduction of an analyte into the medium.

20. The apparatus according to claim 19, wherein the analyte is at least one material selected from the group consisting of pharmaceutical agent, drug, environmental factor, toxin, chemical agent, biological agent, and virus.

21. The apparatus according to claim 15, wherein the means for monitoring detects the degree to which the individual cells adhere to the respective surfaces of particular microelectrodes on the basis of cell/substrate seal resistance.

22. The apparatus according to claim 1, wherein the means for selectively applying an electrical signal includes a multiplexer for selectively applying a signal to each of the microelectrodes.

23. The apparatus according to claim 1, wherein the detecting means includes means for homodyne detection of the resulting signal.

24. The apparatus according to claim 23, wherein the detecting means includes means for multiplying the resulting signals in quadrature with the applied signal and low pass filtering the product to obtain real and imaginary components which are processed by the monitoring means.

25. The apparatus according to claim 1, wherein the characteristics are monitored as the applied electrical signal is varied at each microelectrode across different frequencies.

26. The apparatus according to claim 1, wherein the characteristics are monitored as the applied electrical signal is varied at each microelectrode across different excitation voltages.

27. The apparatus according to claim 1, wherein the means for detecting includes means for heterodyne detection of the resulting signal.

28. The apparatus according to claim 27, wherein the means for heterodyne detection include:

a local oscillator which produces a signal having a frequency based on the frequency of the applied signal and an intermediate frequency, means for multiplying the resulting signal in quadrature by the signal from the local oscillator, and a bandpass filter tuned to the intermediate frequency to filter the product thereof to obtain real and imaginary components of the resulting signal.

29. The apparatus according to claim 1, wherein the means for monitoring includes means for performing spectral analysis of the resulting signal, whereby changes in the characteristics are monitored in real time.

30. An apparatus comprising:

a monolithic device that includes an array of planar microelectrodes disposed on a substrate, the array of microelectrodes being connected to a conductive pattern having contacts points connected thereto;

a cell culture chamber in which the monolithic device is disposed, the cell culture chamber being configured to contain a medium for cultured cells in a volume surrounding the respective surfaces of the microelectrodes, wherein cells are introduced into the cell culture chamber and a portion of the cells adhere to respective surfaces of the microelectrodes, wherein the diameter of a cell is larger than the diameter of a microelectrode;

a reference electrode disposed within the cell culture chamber;

means for selectively applying an electrical signal between each microelectrode and the reference electrode;

means for detecting signals resulting from the application of the signal between the microelectrodes and the reference electrode;

means for processing the detected signals across a spectrum of frequencies to calculate the impedance spectrum of each microelectrode.